The focus of our research is to investigate the hypothesis that amino-carbonyl reactions from ascorbic acid play a role in nuclear sclerosis of the aging lens. On the other hand, recent evidence also suggests oxidant stress from low glutathione (GSH) levels is important in the formation of age-related nuclear cataract (ARNC). Toward testing these hypotheses, we made a number of important breakthroughs in the past five years which include 1) the creation of the hSVCT2 mouse which expresses humanized levels of ascorbic acid in the lens, 2) the demonstration that within 12 mos the lenses turn yellow and accumulate all ascorbylation products present in the human lens, 3) the discovery that both oral and topical L-arginine is a potent inhibitor of crystallin damage in vivo, 4) the successful development of the LEGSKO (lens GSH synthesis conditional knockout) mouse with GSH levels mimicking the low levels in the human lens nucleus, and 5) the identification of arginine residues as the major form of damage in the aging lens. In the coming five years we propose to go full circle and test the two-hit hypothesis that ARNC requires both carbonyl and oxidant stress to form, Specifically, Aim 1 will test the hypothesis that 1) carbonyl attack onto arginine residues of human and mouse gamma-crystallins results in altered protein charge, deguanylation into ornithine at identical sites, protein unfolding, exposure of SH groups, aggregation, and increased interaction with 1-crystallins 2) aggregation occurs at sites prone to opacification, and 3) these changes are preventable with L-arginine in vitro and in the hSVCT2 mouse. Aim 2 will define the role of low lenticular GSH levels in ARNC. Crystallin targets, nature and site of crystallin oxidation and their role in protein aggregation will be determined in vitro and in the novel LEGSKO mouse using a proteomics approach. Glutathione mimetics and pharmacological antioxidants will be tested for their ability to prevent opacification in vivo. Throughout these experiments, the results obtained from investigation in the mouse lens will be compared with changes affecting the crystallins from old normal and ARN cataractous lenses. Aim3 will be to hybridize the hSVCT2 with the LEGSKO mouse to create a mouse with combined carbonyl and oxidant stress, which we hypothesize will facilitate protein aggregation and crosslinking as in the old human lens. Kinetics of protein aggregation and lenticular opacification will be determined compared to control strains, and pharmacological scavengers of carbonyl and oxidant stress will be tested. This approach, we hope, will form the basis for the development of low cost agents for the clinical prevention of human ARNC. To achieve these goals we will use powerful techniques that combine laser capture microdissection, proteomics and dynamic light scattering (DLS). PUBLIC HEALTH RELEVANCE: Investigation into the mechanisms and prevention of age-related nuclear cataract has been hampered by the lack of appropriate animals models. The two major forms of damage to the lens proteins, we hypothesize, stem from damaging vitamin C oxidation products and low antioxidant defense. We now created two humanized mouse models of the aging lens, i.e. one that has humanized levels of vitamin C oxidation products, and one that has low antioxidant defense. Using these models we will study how the damage occurs and leads to cataract, and develop drugs to prevent it.